Analysis of cross-bridge structures between the nuclear membrane and cytoplasmic microtubules.

在盘状盘基钢的聚集阶段，依赖cAMP的信号转导系统变得活跃。我们最近发现，在这一阶段外核膜和细胞质微管（MTs）之间形成了交叉新娘结构。为了寻找构成这些结构的分子，我们检测了盘状田鼠的微管相关蛋白（MAPs）。虽然没有特定于聚集细胞的条带，但在SDS-PAGE上检测到几个共同的成分，42、52、55、59、72、75、95,220和280 kDa条带。用免疫印迹法对各组分进行分析，并测定其部分氨基酸序列。(1) 55kda条带来源于解聚的猪微管蛋白。(2) 42 kDa和95 kDa分别是肌动蛋白和肌动蛋白交联蛋白α肌动蛋白。75 kDa组分的部分氨基酸序列与肌动蛋白封盖蛋白HSC70相似。(3)免疫阴性染色检测与盘基骨柱细胞培养液孵育的MTs组分。抗肌动蛋白抗体的信号存在于附着在MTs末端的结构上。如果这是+端，从细胞质膜下MTs和肌动蛋白系统关系的角度来看，肌动蛋白与MTs的这种结合是有意义的。(4) 280 kDa条带部分氨基酸序列与人细胞角蛋白9一致。由于该条带具有可重复性，且角蛋白分子量为64 kDa，因此该条带似乎不是单纯来源于实验中人体碎片的污染。(5)对其他乐队知之甚少。通过对凝胶上分离的每个带使用抗体检查细胞内分布，将有可能确定组成交叉桥结构的分子

At the aggregation stage of Dictyostelium discoideum, the signal transduction system depending on cAMP becomes active. We recently have found that cross-bride structures are formed between the outer nuclear membrane and cytoplasmic microtubules (MTs) at this stage. To search molecules composing these structures, microtubule-associated proteins (MAPs) of D.discoideum were examined.Although there were no bands specific to aggregating cells, several common components, 42,52,55,59,72,75,95,220 and 280 kDa bands, were detected on SDS-PAGE.Each component was analyzed with western blotting and their partial amino acid sequences also were examined.(1) The 55 kDa band is originated from depolymerized porcine tubulin.(2) The 42 and 95 kDa are actin and an actin-cross linking protein, alpha actinin, respectively. Partial amino acid sequence of the 75 kDa component is similar to HSC70, actin-capping protein.(3) MTs fraction incubated with Dictyostelium cell sup has been examined by immuno-negative staining. Signals for anti-actin antibody are found on structures attached to the terminal end of MTs. If this is the+end, this type of binding of actin to MTs is interested from a point of view of the relation of MTs and actin systems under the cytoplasmic membranes.(4) Partial amino acid sequence of the 280 kDa band coincides with human cytokeratin 9. Since this band reproducibly is found and molecular weight of the keratin is 64 kDa, this band seems not to be originated simply from contaminations of human debris during experiments.(5) Little is known about other bands. It will be possible to determine molecules composing cross-bridge structures through the examination of intracellular distribution using antibody against each band separated on the gels